Molecular Diagnostic Methods

Introduction

p   Molecular methods

n   DNA probes

n   Nucleic acid hybridization

n   Amplification techniques

Advantages and disadvantages

p     Advantages:

n    Test is more specific, sensitive and accurate

n    results are more rapid

p   better monitoring and treatment of diseases.

n    especially useful for:

p   Fastidious microorganisms

p   Hard to detect/culture microbes

p   Characterization of antimicrobial resistance gene

n    Commercial kits

p   Provide a degree of standardization and ease of use

p     Disadvantage:

n    Sensitivity

p   contamination could results in false-positive or false-negative results

p   Requires good clinical laboratory practice, quality assurance measures and experimental controls to be used

Restriction enzymes

p    Naturally occurring enzymes that cut DNA into fragments

n    Cut in predictable and controllable manner

p    Generate pieces of DNA called restriction fragments

n    These fragments can be joined to new fragments

p   Enzymes produce jagged cuts called sticky ends

§    Ends anneal together to form new strand

n    DNA ligase covalently joins fragments

Primers

n   Single-stranded DNA fragments that bind (anneal to) sequences of DNA

n   Used in in vitro DNA synthesis

p  Primer serves as fragment for addition of DNA nucleotides (3’-OH end)

Gel electrophoresis

n   Used to separate DNA fragments according to size

n   Gel must be stained to view DNA

p  Stained with ethidium bromide solution

DNA sequencing

p   Knowing DNA sequence of particular cell helps identify genetic alterations

n   Alterations that may result in disease

p  Sickle cell anemia

§    Due to single base-pair change in gene

p  Cystic fibrosis

§    Caused by three base-pair deletion

p   Human Genome Project

n    Nucleotides have been sequenced

n    may provide diagnostics and treatments for genetic diseases

p    Sanger Method (Dideoxynucleotide chain termination)

n    Use chemically altered nucleotides

p   Dideoxynucleotides

§    Like deoxynucleotide counterparts but lack 3’ OH

§    Incorporation causes chain termination

n    Elements for termination reaction include

p   Single stranded DNA template

p   Primer that anneal to template

p   DNA polymerase

p   4 reactions each containing

"G" tube: All four dNTPs, one is labeled, plus ddGTP
"A" tube: All four dNTPs, one is labeled, plus ddATP
"T" tube: All four dNTPs, one is labeled, plus ddTTP
"C" tube: All four dNTPs, one is labeled, plus ddCTP

 

n    Special gel electrophoresis used to separate DNA fragments by size

p     Automated DNA sequencing

n    Most automated systems use fluorescent dyes to detect newly synthesized DNA

n    Gel electrophoresis used to separate fragments into colored bands

n    Laser used to detect color differences

p   Order of color reflects nucleotide sequence

DNA probes

p    Used to locate nucleotide sequences in DNA or RNA

p    single-stranded piece of DNA tagged with detectable marker

p    will hybridize to complementary fragment of interest

p    Commercial probes available for some pathogens:

n    Species-specific rRNA sequences

n    M. tuberculosis, N. gonorrhoeae, S. aureus, E. coli, H. influenzae, L. monocytogenes, among others

p   Variety of technologies employ DNA probes

n   Colony blotting

n   Southern blotting

n   Fluorescence in situ hybridization (FISH)

n   DNA microarray

Southern blot

p    Uses probes to detect DNA sequences in restriction fragments separated using gel electrophoresis

p    Application = locating DNA sequences similar to ones being studied

p    Genetic screening

n    Identification of mutant genes

n    Cystic fibrosis

n    Genes for Breast cancer and Huntington’s disease

Fluorescence in situ hybridization (FISH)

p    Uses fluorescently labeled probe to detect certain nucleotide sequences

n    Detects sequences inside intact cell

n    Determines:

p   Identity

p   Abundance

p   Relative activity (mRNA)

p    Specimen viewed using fluorescence microscope

n    Mycobacterium tuberculosis in sputum sample

DNA microarray technologies

p    Microarray:

n    tool for analyzing gene expression

n    DNA arrays

p   solid supports (small membrane or glass slide) with fixed patterns of single-stranded DNA fragments attached

n    Enable researchers to screen sample for numerous sequences simultaneously

n    Maps patterns of gene expression

p   Breast cancer

p   DNA chip:

n   Screen a sample for multiple pathogens or expression of many genes

n   primers from different organisms can be used

n   Tagged DNA or mRNA will bind only to its complementary DNA on the chip

n   Bound DNA detected by its fluorescent dye and analyzed by a computer

•          Prepare DNA chip using chosen target DNAs.

•          Generate hybridization solution containing mixture of fluorescently labeled cDNAs.

•          Incubate hybridization mixture containing fluorescently labeled cDNAs with DNA chip.

•          Detect bound cDNA using laser technology and store data in a computer.

•          Analyze data using computational methods.

Western blot

p   Separate proteins by electrophoresis on polyacrylamide gel (by size)

p   Specific proteins can be detected by their reactions with their specific antibodies

Pulse-field gel electrophoresis

p     DNA fingerprinting

n    Use to identify bacterial and viral pathogens

n    Used to track spread of an infectious disease

p     cut chromosomal DNA with a restriction enzyme.

n    resultant fragments too large for conventional electrophoresis (10-800 kb)

p   separated in electrical fields that switch the angle of migration or briefly invert the field.

n    optimal typing method

p   epidemiologically related organism will have predictably similar band profiles if they were derived from a common parent organism.

p     Figure:

n    Bacterial isolates from an outbreak of E. coli O157:H7 in apple juice

Polymerase Chain Reaction (PCR)

p    Creates millions of copies of given region of DNA

n    uses synthetic primers (oligonucleotides) flanking target nucleic sequence of interest

p   Allows for selective replication of chosen regions

§    Termed target DNA

p   Large amounts of DNA can be produced from very small sample

p    Detection and identification of the PCR product by:

n    agarose gel electrophoresis

n    hybridization with a specific oligonucleotide probe,

n    restriction enzyme analysis

n    DNA sequencing.

p    Advantages of PCR:

n    Extremely high sensitivity

n    Easy to set up

n    Fast turnaround time

p    Disadvantages of PCR

n    Extremely liable to contamination

p   with external source of target DNA à false-positive test results

n    High degree of operator skill required

n    A positive result may be difficult to interpret

p   with latent viruses

p     Very useful for identifying pathogens that cannot be cultured

p     PCR determined:

n    Cause of Whipple’s disease (1992)

p   previously an unknown bacterium

p   Causes gastrointestinal and nervous system disorders

p   PCR only reliable method of diagnosing disease

n    Hantavirus as cause of hemorrhagic fever (1993)

p     Commercial kits:

n    Has served to optimize user acceptability

n    Prevent contamination

n    Standardize reagents and testing conditions

n    Makes automation a possibility